{"gene":"NEDD4L","run_date":"2026-06-10T05:19:52","timeline":{"discoveries":[{"year":2001,"finding":"SGK1 phosphorylates Nedd4-2 on Ser444 (and to a lesser extent Ser338) via a PY motif-dependent interaction, reducing the interaction between Nedd4-2 and ENaC and thereby increasing ENaC cell surface expression.","method":"Expression studies in Xenopus laevis oocytes, co-immunoprecipitation, phosphorylation mapping","journal":"The EMBO journal","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal interaction mapping, phosphosite identification, functional readout (ENaC surface expression), independently replicated in a concurrent study (PMID:11696533)","pmids":["11742982"],"is_preprint":false},{"year":2001,"finding":"SGK1 binds to hNedd4-2 via a PY motif in SGK1 and phosphorylates hNedd4-2 (but not hNedd4), reducing hNedd4-2 binding to αENaC and thereby relieving hNedd4-2-mediated inhibition of Na+ absorption.","method":"Co-immunoprecipitation, in vitro phosphorylation assay, electrophysiology in Xenopus oocytes","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — reciprocal Co-IP, in vitro kinase assay, functional rescue, replicated by PMID:11742982","pmids":["11696533"],"is_preprint":false},{"year":2001,"finding":"KIAA0439 (NEDD4L/Nedd4-2) WW domains bind all three ENaC subunits (α, β, γ) via PPxY motifs; a recombinant dominant-negative WW domain construct interferes with Na+-dependent feedback inhibition of ENaC in whole-cell patch-clamp experiments.","method":"GST pulldown, whole-cell patch-clamp electrophysiology","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct binding and functional electrophysiology in single study","pmids":["11244092"],"is_preprint":false},{"year":2002,"finding":"Only WW3 and WW4 domains of Nedd4-2 are required for both binding to ENaC subunits and regulation of Na+ feedback control; both domains together are essential for in vivo function, indicating they interact with distinct non-interchangeable sites in ENaC.","method":"Surface plasmon resonance binding kinetics, whole-cell patch-clamp in mammalian cells, Xenopus oocyte expression","journal":"FASEB journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — two orthogonal methods (binding kinetics + electrophysiology) in single laboratory","pmids":["12424229"],"is_preprint":false},{"year":2005,"finding":"14-3-3 proteins bind directly to SGK1-phosphorylated Nedd4-2 at Ser-468, preserving the activity of SGK1-stimulated ENaC-dependent Na+ current; disrupting the 14-3-3/Nedd4-2 interaction decreases ENaC surface density, likely by allowing Nedd4-2-mediated ubiquitination and ENaC degradation.","method":"Co-immunoprecipitation, Xenopus oocyte electrophysiology, surface biotinylation","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — direct interaction mapping with phosphosite, functional readout, replicated with structural detail in PMID:16716084 and PMID:34294877","pmids":["15677482"],"is_preprint":false},{"year":2006,"finding":"14-3-3 proteins inhibit the interaction between WW domains of hNedd4-2 and the PY motif of ENaC in a manner dependent on SGK1-catalyzed phosphorylation of Ser468 (and similarly PKA-mediated phosphorylation), shielding phosphorylated Nedd4-2 from dephosphorylation.","method":"Surface plasmon resonance spectrometry, Xenopus oocyte electrophysiology","journal":"Biochemistry","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — surface plasmon resonance quantitative binding assay plus functional oocyte data, consistent with prior study","pmids":["16716084"],"is_preprint":false},{"year":2007,"finding":"Nedd4-2 catalyzes ubiquitination of α-, β-, and γENaC subunits at the cell surface; monoubiquitination is sufficient to reduce ENaC surface expression and current; HECT domain mutation abolishes activity; Nedd4-2 selectively reduces surface ENaC without affecting immature biosynthetic ENaC.","method":"Ubiquitination assays, surface biotinylation, siRNA knockdown, overexpression, electrophysiology","journal":"The Journal of biological chemistry","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (ubiquitination assay, biotinylation, electrophysiology, siRNA/overexpression) in rigorous single study","pmids":["17502380"],"is_preprint":false},{"year":2008,"finding":"The WW domains of Nedd4-2 bind an intramolecular PY motif (LPXY) within its own HECT domain, inhibiting auto-ubiquitination and stabilizing Nedd4-2; mutation of this HECT PY motif decreases Nedd4-2 stability without affecting ENaC ubiquitination; ENaC binding disrupts this intramolecular interaction, triggering Nedd4-2 self-ubiquitination and degradation.","method":"Pulse-chase experiments, co-immunoprecipitation, mutational analysis, ubiquitination assays","journal":"The Biochemical journal","confidence":"High","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (pulse-chase, Co-IP, mutagenesis, ubiquitination assay) in single study revealing a novel autoinhibitory mechanism","pmids":["18498246"],"is_preprint":false},{"year":2004,"finding":"GRK2 phosphorylates the C terminus of the ENaC β subunit and renders ENaC insensitive to inhibition by Nedd4-2, maintaining channels in the active state; this represents a mechanism by which GRK2 counteracts Nedd4-2-dependent channel inhibition.","method":"Xenopus oocyte electrophysiology, in vitro kinase assay, epistasis experiments","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — in vitro kinase assay plus functional oocyte electrophysiology in single study","pmids":["15284439"],"is_preprint":false},{"year":2007,"finding":"GRK2 physically interacts with both Nedd4 and Nedd4-2, and phosphorylates both ligases at multiple sites including Thr466 in the WW3 domain region of Nedd4.","method":"Co-immunoprecipitation, in vitro phosphorylation assay","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP and in vitro kinase assay in single study, extends mechanistic understanding from PMID:15284439","pmids":["17544362"],"is_preprint":false},{"year":2011,"finding":"Knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung, causing premature fetal lung fluid clearance and perinatal lethality; surviving knockouts develop lethal sterile lung inflammation, establishing Nedd4-2 as an essential regulator of ENaC-dependent lung fluid clearance in vivo.","method":"Genetic knockout mouse model, electrophysiology, immunoblotting","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — clean in vivo knockout with defined molecular and physiological phenotype, multiple orthogonal readouts","pmids":["21505443"],"is_preprint":false},{"year":2011,"finding":"Nedd4-2 co-immunoprecipitates with NCC and stimulates NCC ubiquitylation at the cell surface; catalytically inactive Nedd4-2 fails to decrease NCC activity; SGK1 prevents this inhibition in a kinase-dependent manner; double phosphorylation of Nedd4-2 at Ser222/Ser328 abolishes SGK1-mediated rescue. Unlike ENaC regulation, this does not require a PY motif in NCC.","method":"Co-immunoprecipitation, ubiquitination assay, Xenopus oocyte electrophysiology, surface biotinylation, knockout mice","journal":"Journal of the American Society of Nephrology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, electrophysiology, KO mice) in single rigorous study","pmids":["21852580"],"is_preprint":false},{"year":2013,"finding":"Inducible, nephron-specific Nedd4-2 knockout in mice causes salt-sensitive hypertension characterized by increased NCC, β/γENaC (but not αENaC) and ROMK expression; thiazide treatment reverses hypertension, demonstrating NCC as a primary in vivo target of NEDD4-2 in the kidney.","method":"Conditional knockout mouse model, immunoblotting, blood pressure telemetry, pharmacological rescue","journal":"The Journal of clinical investigation","confidence":"High","confidence_rationale":"Tier 2 / Strong — inducible cell-type-specific KO with defined molecular phenotype and pharmacological rescue, multiple independent readouts","pmids":["23348737"],"is_preprint":false},{"year":2014,"finding":"Nedd4-2 regulates voltage-gated sodium channel (Nav) activity specifically in response to elevated intracellular Na+ in fetal cortical neurons but does not affect steady-state Nav activity, as shown using Nedd4-2-deficient mice; no such regulation was observed in dorsal root ganglia neurons from the same mice.","method":"Nedd4-2 knockout mouse neurons, electrophysiology, intracellular Na+ manipulation","journal":"The Biochemical journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — clean KO model with defined electrophysiological phenotype in specific cell type, single laboratory","pmids":["24152020"],"is_preprint":false},{"year":2016,"finding":"NEDD4-2 ubiquitinates TRAF3 at Cys56 and Cys124 (zinc finger residues) via K29-linked cysteine ubiquitination; this promotes enhanced TRAF3 association with K48/K63 E3 ligases (cIAP1/2, HECTD3) and TRAF3-TBK1 interaction, leading to TBK1 and IRF3 phosphorylation and type I interferon production upon virus infection.","method":"Co-immunoprecipitation, ubiquitination assay, mass spectrometry, site-directed mutagenesis, knockout cells and mice","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including MS, mutagenesis, Co-IP, KO in vitro and in vivo in single rigorous study","pmids":["33608556"],"is_preprint":false},{"year":2017,"finding":"Nedd4-2 ubiquitinates and downregulates the cardiac potassium channel hERG1 via a PY motif in the C-terminus of hERG1; Nedd4-2 co-expression decreases total and surface hERG1 protein and whole-cell current; deletion of the PY motif abrogates Nedd4-2-dependent regulation.","method":"Co-immunoprecipitation, surface biotinylation, Western blot, electrophysiology in HEK293 cells","journal":"Journal of molecular and cellular cardiology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — reciprocal Co-IP, biotinylation, and electrophysiology in single lab study","pmids":["21463633"],"is_preprint":false},{"year":2017,"finding":"Nedd4-2 ubiquitinates and decreases surface expression and activity of organic anion transporter OAT3; this requires the WW3 and WW4 domains of Nedd4-2; a physical interaction between OAT3 and Nedd4-2 was confirmed in cultured cells and rat kidney slices.","method":"Co-immunoprecipitation, ubiquitination assay, transport activity assay, siRNA knockdown, mutagenesis","journal":"Molecular pharmaceutics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (Co-IP, ubiquitination assay, transport assay, mutagenesis) in single laboratory","pmids":["26651153"],"is_preprint":false},{"year":2017,"finding":"NEDD4L binds to and promotes ubiquitination and proteasome-dependent degradation of PIK3CA (p110α catalytic subunit of PI3K), negatively regulating PIK3CA protein levels and affecting PI3K-AKT signaling; NEDD4L does not ubiquitinate the PI3K regulatory subunit.","method":"Co-immunoprecipitation, ubiquitination assay, cycloheximide chase, overexpression/knockdown with immunoblotting","journal":"The Journal of biological chemistry","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple biochemical methods in single laboratory","pmids":["27339899"],"is_preprint":false},{"year":2017,"finding":"Nedd4-2 ubiquitinates phosphorylated Syk (a tyrosine kinase essential for FcɛRI signaling) with adaptor protein Ndfip1, limiting the intensity and duration of IgE-FcɛRI signaling in mast cells; loss of Nedd4-2 or Ndfip1 in mast cells causes exacerbated cutaneous anaphylaxis in vivo.","method":"Co-immunoprecipitation, ubiquitination assay, mast cell-specific knockout mice, in vivo anaphylaxis model","journal":"Nature communications","confidence":"High","confidence_rationale":"Tier 2 / Strong — cell-type-specific KO with in vivo phenotype, biochemical validation of substrate ubiquitination, single rigorous study","pmids":["27786273"],"is_preprint":false},{"year":2018,"finding":"GPCR PAR1 stimulates c-Src-mediated tyrosine phosphorylation of NEDD4-2 at Y485 (within the linker between WW2 and WW3 domains), activating NEDD4-2 E3 ligase activity and driving p38 signaling and endothelial barrier disruption; Y485 mutation impairs E3 ligase activity and p38 activation.","method":"Mass spectrometry phosphoproteomics, site-directed mutagenesis, kinase inhibitor experiments, endothelial permeability assays","journal":"Cell reports","confidence":"High","confidence_rationale":"Tier 1-2 / Moderate — MS phosphosite identification, mutagenesis, functional rescue in single rigorous study","pmids":["30232011"],"is_preprint":false},{"year":2018,"finding":"Nedd4-2 binds to Kir5.1 via a phosphothreonine (TPVT) motif at its C-terminus; this interaction facilitates ubiquitination of the Kir4.1/Kir5.1 heterotetramer (not Kir4.1 alone) and reduces Kir4.1 expression and K+ currents in the distal convoluted tubule.","method":"Co-immunoprecipitation, GST pulldown, ubiquitination assay, patch-clamp electrophysiology, Kir5.1 mutagenesis, kidney-specific Nedd4-2 knockout mice","journal":"American journal of physiology. Renal physiology","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods including KO mice, mutagenesis, electrophysiology, pulldown and ubiquitination assays","pmids":["29897283"],"is_preprint":false},{"year":2019,"finding":"NEDD4L binds ULK1 in pancreatic cancer cells and promotes its ubiquitination and degradation; NEDD4L depletion stabilizes ULK1, enhancing autophagy activity and mitochondrial metabolic function; NEDD4L also regulates the glutamine transporter ASCT2.","method":"Co-immunoprecipitation, siRNA knockdown, immunoblotting, oxygen consumption rate measurement, xenograft mouse models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, knockdown with functional readout, in vivo xenograft, single laboratory","pmids":["31959741"],"is_preprint":false},{"year":2019,"finding":"NEDD4 and NEDD4L negatively regulate Wnt/β-catenin signaling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation; loss of both ligases enhances ISC proliferation, increases RSPO sensitivity, and accelerates tumor development in Apcmin mice.","method":"Conditional knockout mice, co-immunoprecipitation, ubiquitination assay, organoid culture, tumor phenotyping","journal":"The EMBO journal","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP and KO with defined phenotype, but involves both NEDD4 and NEDD4L making NEDD4L-specific contribution partially confounded","pmids":["31867777"],"is_preprint":false},{"year":2020,"finding":"NEDD4L identifies and promotes ubiquitination and proteasomal degradation of lactotransferrin (LTF) via direct protein-protein interaction, thereby inhibiting intracellular iron accumulation and ferroptotic cell death.","method":"siRNA screen, mass spectrometry for substrate identification, co-immunoprecipitation, ubiquitination assay, ferroptosis cell death assays","journal":"Biochemical and biophysical research communications","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — MS-based substrate identification, Co-IP, functional ferroptosis assay in single laboratory","pmids":["32811647"],"is_preprint":false},{"year":2020,"finding":"NEDD4L ubiquitinates and promotes degradation of SMAD2, a TGFβ pathway transcription modulator; USP7 deubiquitinates NEDD4L (stabilizing it), and this USP7-NEDD4L-SMAD2 axis controls autophagy and protein quality control in a neuroprotective pathway conserved from C. elegans to mammals.","method":"Genetic epistasis in C. elegans and Drosophila, co-immunoprecipitation, ubiquitination assay in mammalian cells","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — cross-species epistasis plus biochemical validation, but mechanistic detail of NEDD4L-SMAD2 interaction assessed in single lab","pmids":["33106424"],"is_preprint":false},{"year":2020,"finding":"NEDD4-2 ubiquitinates α-synuclein after focal ischemia; Nedd4l knockout reduces post-ischemic poly-ubiquitinated α-synuclein and exacerbates ischemic brain damage; co-immunoprecipitation confirms enhanced α-synuclein–Nedd4l interaction after stroke.","method":"Transient MCAO mouse model, knockout mice, co-immunoprecipitation, behavioral testing","journal":"Journal of cerebral blood flow and metabolism","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — KO mouse with defined phenotype, Co-IP for interaction, single laboratory","pmids":["32703111"],"is_preprint":false},{"year":2020,"finding":"NEDD4-2 mediates ubiquitination and degradation of Merlin/NF2 (mostly mono/di-ubiquitination at K396); this requires the scaffold protein AMOTL1 to bridge NEDD4L to Merlin; ubiquitination is promoted by Ser518 dephosphorylation in response to Ca2+ signaling or cell detachment, and is required for Merlin binding to Lats1 and Hippo pathway activation.","method":"Co-immunoprecipitation, ubiquitination assay, mutagenesis, Ca2+ signaling experiments, Lats1 kinase activity assay","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods (Co-IP, ubiquitination, mutagenesis, kinase assay) in single laboratory","pmids":["33058421"],"is_preprint":false},{"year":2017,"finding":"Nedd4-2 ubiquitinates the SP-C proprotein at K6 (via its PY motif binding to Nedd4-2 WW domains), and this ubiquitination is required for lumenal relocation of SP-C within multivesicular bodies, leading to SP-C processing and secretion.","method":"GST pulldown with recombinant proteins, co-immunoprecipitation, ubiquitination assay, alanine mutagenesis, adenoviral infection in type II cells","journal":"American journal of respiratory cell and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (pulldown, Co-IP, ubiquitination, mutagenesis, functional secretion assay) in single laboratory","pmids":["19423771"],"is_preprint":false},{"year":2017,"finding":"NDFIP2 (and NDFIP1) serve as adaptors that bridge NEDD4/NEDD4L to AQP2, enabling NEDD4/NEDD4L-mediated ubiquitination and degradation of AQP2; NEDD4 and NEDD4L do not bind AQP2 directly.","method":"Membrane Yeast Two-Hybrid, siRNA knockdown, co-immunoprecipitation, ubiquitination assay, immunoblotting in HEK293 and mpkCCD cells","journal":"PloS one","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple methods identifying indirect substrate engagement mechanism; single laboratory","pmids":["28931009"],"is_preprint":false},{"year":2017,"finding":"Nedd4-2 ubiquitinates epilepsy-associated GluA1 subunit of AMPA receptors, reducing GluA1 surface expression; epilepsy-associated missense mutations in Nedd4-2 disrupt GluA1 ubiquitination; genetic reduction of GluA1 normalizes seizure susceptibility in Nedd4-2andi mice.","method":"Knockout/knock-in mouse model, ubiquitination assay, surface biotinylation, extracellular electrophysiology, in vivo seizure model, genetic epistasis","journal":"PLoS genetics","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple orthogonal methods plus in vivo genetic epistasis rescue, rigorous mechanistic study","pmids":["28212375"],"is_preprint":false},{"year":2021,"finding":"14-3-3 protein binding to phosphorylated Ser342 and Ser448 of Nedd4-2 induces a structural rearrangement that inhibits intramolecular interactions between the HECT and WW domains, providing the structural basis for 14-3-3-mediated Nedd4-2 regulation.","method":"Integrative structural biology (SAXS, NMR, HDX-MS), mutagenesis, phosphopeptide binding assays","journal":"Communications biology","confidence":"High","confidence_rationale":"Tier 1 / Moderate — integrative structural biology plus functional validation, multiple orthogonal biophysical methods in single study","pmids":["34294877"],"is_preprint":false},{"year":2019,"finding":"PLPP/CIN (pyridoxal-5'-phosphate phosphatase/chronophin) specifically dephosphorylates NEDD4-2 at Ser448, reducing Nedd4-2 ubiquitination and facilitating NEDD4-2-mediated ubiquitination of GluA1, thereby modulating neuronal excitability.","method":"In vitro phosphatase assay, co-immunoprecipitation, ubiquitination assay, electrophysiology, knockout cell models","journal":"Cell death & disease","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — direct phosphatase assay plus functional ubiquitination and electrophysiology in single laboratory","pmids":["31320629"],"is_preprint":false},{"year":2022,"finding":"NEDD4L directly interacts with MEKK2 and promotes its poly-ubiquitination and degradation; IL-17-induced Ser520 phosphorylation of MEKK2 is required for NEDD4L-mediated MEKK2 degradation and shutdown of IL-17R signaling; Nedd4l-deficient mice show increased susceptibility to IL-17-induced inflammation.","method":"Co-immunoprecipitation, ubiquitination assay, mutagenesis, knockout mice, experimental autoimmune encephalomyelitis (EAE) model","journal":"EMBO reports","confidence":"High","confidence_rationale":"Tier 2 / Strong — multiple methods (Co-IP, ubiquitination, mutagenesis, KO mice with in vivo disease model), rigorous single-laboratory study","pmids":["36161689"],"is_preprint":false},{"year":2022,"finding":"NEDD4L directly interacts with GP130 and mediates its Lys-27-linked ubiquitination and proteasomal degradation, negatively regulating IL-6/GP130/STAT3 signaling in keratinocytes and suppressing skin hyperplasia.","method":"Co-immunoprecipitation, ubiquitination assay, mutagenesis, knockout mice (imiquimod skin model), immunohistochemistry","journal":"EMBO reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, and KO mice with phenotypic readout in single laboratory","pmids":["33769697"],"is_preprint":false},{"year":2022,"finding":"NEDD4L directly interacts with GPX4 and promotes its ubiquitination and degradation, facilitating ferroptosis in granulosa cells.","method":"Co-immunoprecipitation, ubiquitination assay, cell viability assay, ferroptosis marker measurement","journal":"Endocrine connections","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and ubiquitination assay, single laboratory, limited mechanistic follow-up","pmids":["36662677"],"is_preprint":false},{"year":2022,"finding":"NEDD4L promotes ubiquitination and degradation of ENO1 (enolase 1), suppressing glycolysis and proliferation in oral squamous cell carcinoma; ENO1 overexpression reverses functional effects of NEDD4L overexpression.","method":"Ubiquitination assay, co-immunoprecipitation, overexpression/knockdown with glycolysis measurements, in vivo tumor model","journal":"Cancer biology & therapy","confidence":"Low","confidence_rationale":"Tier 3 / Weak — computational substrate prediction followed by single Co-IP and ubiquitination assay in single laboratory","pmids":["35316145"],"is_preprint":false},{"year":2023,"finding":"NEDD4L mediates ubiquitination and degradation of YBX1 protein when recruited by circNEIL3; direct interaction between NEDD4L and YBX1 was confirmed, and this promotes proteasomal degradation of YBX1 to suppress tumor metastasis.","method":"Co-immunoprecipitation, ubiquitination assay, loss-of-function/overexpression experiments in cancer cells and in vivo","journal":"Proceedings of the National Academy of Sciences of the United States of America","confidence":"Low","confidence_rationale":"Tier 3 / Weak — mechanistic finding about NEDD4L-YBX1 interaction is secondary to the circRNA study; single lab, single Co-IP","pmids":["36961927"],"is_preprint":false},{"year":2023,"finding":"NEDD4L ubiquitinates and degrades ACSL4, a key ferroptosis regulator; YAP facilitates NEDD4L transcription, so YAP→NEDD4L→ACSL4 ubiquitination axis reduces ferroptosis in myocardial ischemia-reperfusion injury.","method":"Co-immunoprecipitation, ChIP, dual-luciferase reporter assay, Western blot, in vivo MIRI model","journal":"The Canadian journal of cardiology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for NEDD4L-ACSL4 interaction, transcriptional mechanism for YAP-NEDD4L included, single laboratory","pmids":["37541340"],"is_preprint":false},{"year":2023,"finding":"14-3-3σ promotes binding of S448-phosphorylated NEDD4L to HIF-1α, enhancing HIF-1α poly-ubiquitination and proteasomal degradation; this suppresses hypoxia-induced tumor progression in colorectal cancer.","method":"Co-immunoprecipitation, ubiquitination assay, mutagenesis, patient sample correlation analysis","journal":"Cell reports","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, mutagenesis in single laboratory with clinical correlation","pmids":["37494179"],"is_preprint":false},{"year":2024,"finding":"NEDD4L ubiquitinates and degrades TFRC (transferrin receptor/CD71), reducing iron accumulation; METTL3-mediated m6A methylation stabilizes NEDD4L mRNA, enhancing NEDD4L expression and the NEDD4L-TFRC axis to inhibit ferroptosis and neuronal damage in ischemic stroke.","method":"Co-immunoprecipitation, ubiquitination assay, half-life experiments, MCAO mouse model, NEDD4L knockout model","journal":"Cell biology and toxicology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, KO model, in vivo MCAO, multiple methods in single laboratory","pmids":["38302612"],"is_preprint":false},{"year":2021,"finding":"Non-SUMOylated CRMP2 forms a complex with Numb, Eps15, and Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7; silencing Nedd4-2 in DRG neurons from CRMP2K374A/K374A mice restores NaV1.7 surface expression and sodium currents.","method":"siRNA knockdown in DRG neurons, electrophysiology, in vivo nerve injury model","journal":"Molecular brain","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — genetic approach in primary neurons with electrophysiological readout; NEDD4L role inferred from complex membership and siRNA data","pmids":["33478555"],"is_preprint":false},{"year":2020,"finding":"NEDD4L interacts with Piezo2 in co-immunoprecipitation experiments in HEK293T cells and inhibits Piezo2 mechanically-activated currents when co-expressed; Nedd4-2 upregulation in hypertensive rat nodose ganglia correlates with Piezo2 downregulation.","method":"Co-immunoprecipitation, electrophysiology in HEK293T, in vivo knockdown, spontaneously hypertensive rat model","journal":"Pharmacological research","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and electrophysiology, correlational in vivo data, single laboratory","pmids":["33352230"],"is_preprint":false},{"year":2023,"finding":"NEDD4L deficiency in intestinal epithelial cells inhibits expression of SLC3A2 (protein level only, not mRNA), reducing GPX4 expression and promoting ferroptosis; this mechanistic axis (NEDD4L→SLC3A2→GPX4) maintains intestinal homeostasis and restricts inflammatory bowel disease.","method":"Conditional and global KO mice, DSS/TNBS colitis and AOM-DSS tumor models, immunoblotting, ferroptosis inhibitor rescue","journal":"The Journal of clinical investigation","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple KO models with defined phenotype and pharmacological rescue; mechanistic link to SLC3A2 post-translational regulation is partially indirect","pmids":["39688910"],"is_preprint":false},{"year":2005,"finding":"Both Nedd4 and Nedd4-2 use UbcH5b as their primary E2 ubiquitin-conjugating enzyme in in vitro ubiquitination assays; Ube2e3 is used less efficiently by both.","method":"In vitro ubiquitination assay with panel of E2 enzymes","journal":"The international journal of biochemistry & cell biology","confidence":"Medium","confidence_rationale":"Tier 1 / Moderate — reconstituted in vitro ubiquitination assay; single laboratory but clean biochemical result","pmids":["16337426"],"is_preprint":false},{"year":2009,"finding":"NEDD4L directly binds surfactant protein-C (SP-C) proprotein via interaction of its WW domain with the PPDY motif in SP-C residues 12-17; NEDD4L ubiquitinates SP-C at K6 (but not K34); this ubiquitination is required for SP-C proprotein processing.","method":"GST pulldown with recombinant proteins, endogenous Co-IP from lung lysates, ubiquitination assay, mutagenesis, SP-C secretion assay","journal":"American journal of respiratory cell and molecular biology","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple orthogonal methods (GST pulldown, endogenous Co-IP, ubiquitination assay, mutagenesis, functional secretion assay) in single laboratory","pmids":["19423771"],"is_preprint":false},{"year":2016,"finding":"NEDD4L overexpression up-regulates autophagy; knockdown suppresses both basal and ER stress-induced autophagy in cultured cells; Nedd4-2 expression is induced by ER stress via IRE1/XBP1s pathway, linking UPR to autophagic response.","method":"Pharmacological ER stress induction, siRNA knockdown, overexpression, IRE1 inhibition, immunoblotting for autophagy markers","journal":"FASEB journal","confidence":"Low","confidence_rationale":"Tier 3 / Weak — functional cell biology without direct substrate identification for autophagy regulation; single laboratory","pmids":["27022162"],"is_preprint":false},{"year":2023,"finding":"Decreased NEDD4L expression leads to impaired TXNIP ubiquitination and its accumulation in NASH liver; NEDD4L-mediated TXNIP degradation is part of a NEDD4L-TXNIP-CHOP axis that controls hepatocyte apoptosis in nonalcoholic steatohepatitis.","method":"Four independent NASH mouse models, gain/loss-of-function studies, co-immunoprecipitation, ubiquitination assay","journal":"Theranostics","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — multiple animal models, Co-IP, ubiquitination assay; NEDD4L-TXNIP interaction mechanistically validated","pmids":["37153733"],"is_preprint":false},{"year":2023,"finding":"NEDD4L mediates ubiquitination and degradation of STAT3, as shown by co-immunoprecipitation in glioma cells; NEDD4L-induced ferroptosis is rescued by forced expression of STAT3, placing STAT3 downstream of NEDD4L in glioma ferroptosis regulation.","method":"Co-immunoprecipitation, ubiquitination assay, genetic rescue experiments, xenograft mouse model","journal":"Journal of oncology","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP and rescue experiment in single laboratory","pmids":["36618071"],"is_preprint":false},{"year":2022,"finding":"NEDD4L binds the 19S proteasome and promotes autophagy; NEDD4L suppression reduces bortezomib sensitivity in multiple myeloma through autophagy inhibition.","method":"Co-immunoprecipitation with proteasome components, knockdown with autophagy and viability assays, in vivo mouse model","journal":"Cell death & disease","confidence":"Low","confidence_rationale":"Tier 3 / Weak — single Co-IP for proteasome binding, functional autophagy data without defined substrate; single laboratory","pmids":["35236820"],"is_preprint":false},{"year":2023,"finding":"NEDD4L ubiquitinates and degrades TβRII (TGF-β receptor II), the first signaling enzyme of TGF-β1 pathway, suppressing lung myofibroblast differentiation; transcription factor E2F inhibits NEDD4L expression, and blocking E2F rescues NEDD4L levels and mitigates experimental pulmonary fibrosis.","method":"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression, E2F inhibitor experiments, animal model of pulmonary fibrosis","journal":"Translational research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo model; single laboratory","pmids":["36257596"],"is_preprint":false},{"year":2023,"finding":"NEDD4L mediates ubiquitination and degradation of ACE2 in neurogenic hypertension; Ang-II-induced upregulation of Nedd4-2 in the brain promotes ACE2 ubiquitination; mutation of lysine residues in the ACE2 C-terminus confers ubiquitination resistance.","method":"Co-immunoprecipitation, ubiquitination assay, mutagenesis, gene therapy in brain regions, blood pressure telemetry","journal":"Cardiovascular research","confidence":"Medium","confidence_rationale":"Tier 2 / Moderate — Co-IP, ubiquitination, mutagenesis, in vivo optogenetics/telemetry; single laboratory","pmids":["37161607"],"is_preprint":false}],"current_model":"NEDD4L (NEDD4-2) is a HECT-type E3 ubiquitin ligase whose WW domains bind PPxY (PY) motifs in substrate proteins to catalyze their ubiquitination, targeting them for endocytosis and/or proteasomal/lysosomal degradation; its best-characterized substrates include the epithelial Na+ channel ENaC, the Na+-Cl- cotransporter NCC, voltage-gated Na+ and K+ channels, AMPA receptor GluA1, and numerous signaling proteins (TRAF3, MEKK2, PIK3CA, LGR5/DVL2, Merlin/NF2, SMAD2, HIF-1α, TβRII, ACE2, and others); its activity is tightly regulated by SGK1-mediated phosphorylation (which recruits 14-3-3 proteins to sterically inhibit WW-PY interactions), by c-Src-mediated tyrosine phosphorylation at Y485 (which activates it downstream of GPCRs), by PLPP/CIN-mediated dephosphorylation, and by an intramolecular autoinhibitory interaction between its own HECT-domain PY motif and its WW domains that is disrupted upon substrate engagement."},"narrative":{"mechanistic_narrative":"NEDD4L (NEDD4-2) is a HECT-type E3 ubiquitin ligase that controls the surface abundance and activity of ion channels, transporters, receptors, and signaling proteins by catalyzing their ubiquitination, with UbcH5b serving as its primary E2 conjugating enzyme [PMID:16337426]. Its WW domains engage PPxY (PY) motifs in substrates—paradigmatically all three ENaC subunits, where WW3 and WW4 contribute distinct non-interchangeable contacts—to drive substrate ubiquitination and endocytic/degradative removal from the cell surface [PMID:11244092, PMID:12424229, PMID:17502380]. NEDD4L activity is gated by a multilayered phosphoregulatory network: SGK1 phosphorylates NEDD4-2 (at Ser444/Ser468 and related sites) to weaken WW-substrate engagement and recruit 14-3-3 proteins, which bind phospho-NEDD4-2 and impose a structural rearrangement that blocks intramolecular HECT-WW contacts and protects the phosphosites from dephosphorylation [PMID:11742982, PMID:11696533, PMID:15677482, PMID:16716084, PMID:34294877]; conversely PLPP/CIN dephosphorylates Ser448 to license substrate ubiquitination [PMID:31320629], and GPCR/c-Src signaling phosphorylates Tyr485 to activate the ligase [PMID:30232011]. An autoinhibitory intramolecular interaction between the WW domains and a PY motif in NEDD4-2's own HECT domain stabilizes the ligase and is relieved upon substrate binding, coupling target engagement to self-ubiquitination [PMID:18498246]. Through these mechanisms NEDD4L is an essential in vivo regulator of renal and pulmonary sodium handling—targeting ENaC, the Na-Cl cotransporter NCC, and Kir4.1/Kir5.1—such that its loss causes salt-sensitive hypertension and defective lung fluid clearance [PMID:21505443, PMID:21852580, PMID:23348737, PMID:29897283]. Beyond epithelial transport, NEDD4L ubiquitinates a broad substrate repertoire to shape neuronal excitability (GluA1, NaV channels) [PMID:28212375, PMID:24152020], innate and adaptive immune signaling (TRAF3, Syk, MEKK2, GP130) [PMID:33608556, PMID:27786273, PMID:36161689, PMID:33769697], and growth/stress pathways (PIK3CA, LGR5/DVL2, Merlin/NF2, SMAD2, TβRII, HIF-1α), often via adaptor- or scaffold-mediated substrate recruitment such as NDFIP1/2 and AMOTL1 [PMID:27339899, PMID:31867777, PMID:33058421, PMID:33106424, PMID:36257596, PMID:37494179, PMID:28931009]. Epilepsy-associated missense mutations in NEDD4-2 disrupt GluA1 ubiquitination and alter seizure susceptibility, linking the ligase to neuronal disease [PMID:28212375].","teleology":[{"year":2001,"claim":"Establishing how a kinase signal is transduced into ion-channel abundance, SGK1 was shown to phosphorylate NEDD4-2 via a PY-motif interaction, weakening NEDD4-2/ENaC binding and raising channel surface expression — defining the core SGK1-NEDD4-2-ENaC regulatory axis.","evidence":"Co-IP, phosphosite mapping, in vitro kinase assay, and electrophysiology in Xenopus oocytes","pmids":["11742982","11696533"],"confidence":"High","gaps":["Did not resolve the structural basis of phospho-dependent WW disengagement","Selectivity for hNedd4-2 over hNedd4 mechanistically unexplained at this stage"]},{"year":2001,"claim":"To define substrate recognition, the WW domains of NEDD4L were shown to bind PPxY motifs in all three ENaC subunits, with a dominant-negative WW construct disrupting Na+ feedback inhibition — establishing direct WW-PY engagement as the recognition mode.","evidence":"GST pulldown and whole-cell patch-clamp","pmids":["11244092"],"confidence":"Medium","gaps":["Catalytic ubiquitination of ENaC not yet demonstrated","Relative contribution of individual WW domains unresolved"]},{"year":2002,"claim":"Refining the recognition map, WW3 and WW4 were shown to be the binding-competent domains contacting distinct non-interchangeable ENaC sites, both required for in vivo function.","evidence":"Surface plasmon resonance kinetics and patch-clamp","pmids":["12424229"],"confidence":"Medium","gaps":["Structural identity of the two distinct ENaC sites not defined","Generality to non-ENaC substrates not tested"]},{"year":2005,"claim":"Explaining how the SGK1 signal is sustained, 14-3-3 proteins were found to bind SGK1-phosphorylated NEDD4-2 at Ser468, preserving stimulated ENaC current — adding a second tier of regulation.","evidence":"Co-IP, oocyte electrophysiology, surface biotinylation","pmids":["15677482","16716084"],"confidence":"High","gaps":["Structural mechanism of 14-3-3 inhibition not yet resolved","Whether 14-3-3 acts on substrates beyond ENaC unknown"]},{"year":2007,"claim":"Confirming catalytic action, NEDD4-2 was shown to ubiquitinate cell-surface ENaC subunits — monoubiquitination sufficing to reduce surface current — with HECT activity required, establishing ubiquitination as the effector step.","evidence":"Ubiquitination assays, biotinylation, siRNA/overexpression, electrophysiology","pmids":["17502380"],"confidence":"High","gaps":["Downstream endocytic/degradative machinery not defined","Ubiquitin chain linkage on ENaC not specified"]},{"year":2008,"claim":"Revealing intrinsic autoregulation, NEDD4-2 WW domains were found to bind an intramolecular HECT PY motif that suppresses auto-ubiquitination and stabilizes the ligase, with substrate binding disrupting this interaction — coupling target engagement to ligase turnover.","evidence":"Pulse-chase, Co-IP, mutagenesis, ubiquitination assays","pmids":["18498246"],"confidence":"High","gaps":["Structural conformation of the autoinhibited state not resolved here","Interplay with 14-3-3 regulation not yet integrated"]},{"year":2011,"claim":"Testing physiological essentiality, Nedd4-2 knockout mice revealed that the ligase is required in vivo for ENaC-dependent fetal lung fluid clearance, with loss causing perinatal lethality and lung inflammation.","evidence":"Genetic knockout mouse, electrophysiology, immunoblotting","pmids":["21505443"],"confidence":"High","gaps":["Tissue-specific contributions not dissected","Adult renal phenotype not addressed in this model"]},{"year":2011,"claim":"Extending substrate scope in the kidney, NEDD4-2 was shown to ubiquitinate NCC at the cell surface in a PY-motif-independent manner, with SGK1 antagonizing this regulation.","evidence":"Co-IP, ubiquitination assay, oocyte electrophysiology, biotinylation, KO mice","pmids":["21852580"],"confidence":"High","gaps":["Recognition mode for the PY-independent NCC interaction undefined","Adaptor involvement not excluded"]},{"year":2013,"claim":"Defining the dominant renal target, nephron-specific inducible Nedd4-2 knockout produced thiazide-reversible salt-sensitive hypertension with elevated NCC, establishing NCC as a primary in vivo kidney substrate.","evidence":"Conditional KO mouse, immunoblotting, blood pressure telemetry, pharmacological rescue","pmids":["23348737"],"confidence":"High","gaps":["Subunit-selective ENaC effects (β/γ but not α) mechanistically unexplained","Relative quantitative contribution of NCC vs ENaC not parsed"]},{"year":null,"claim":"Broadening to channels, transporters, and signaling, NEDD4L was shown across studies to ubiquitinate Nav channels, hERG1, OAT3, Kir4.1/Kir5.1, GluA1, SP-C, AQP2, and to regulate immune and growth pathways via TRAF3, Syk, MEKK2, GP130, PIK3CA, LGR5/DVL2, Merlin/NF2, SMAD2, TβRII, HIF-1α and ferroptosis regulators.","evidence":"Co-IP, ubiquitination assays, mutagenesis, KO mice, and disease models across multiple substrates","pmids":["24152020","21463633","26651153","29897283","28212375","44","28931009","33608556","27786273","36161689","33769697","27339899","31867777","33058421","33106424","36257596","37494179"],"confidence":"Medium","gaps":["Many substrate interactions rest on single-laboratory Co-IP/ubiquitination data","Ubiquitin chain linkages and adaptor requirements differ across substrates and are incompletely mapped"]},{"year":2018,"claim":"Identifying an activating modification, GPCR-driven c-Src phosphorylation of NEDD4-2 at Tyr485 was shown to switch on E3 ligase activity and downstream p38 signaling — a regulatory input opposite to inhibitory phosphorylation.","evidence":"MS phosphoproteomics, mutagenesis, kinase inhibitors, endothelial permeability assays","pmids":["30232011"],"confidence":"High","gaps":["Direct substrate of the activated ligase in this context not fully defined","Structural effect of Y485 phosphorylation unresolved"]},{"year":2021,"claim":"Providing the structural basis of inhibition, integrative structural biology showed 14-3-3 binding to phospho-Ser342/Ser448 rearranges NEDD4-2 to block intramolecular HECT-WW contacts, unifying the phospho/14-3-3 and autoinhibition models.","evidence":"SAXS, NMR, HDX-MS, mutagenesis, phosphopeptide binding","pmids":["34294877"],"confidence":"High","gaps":["High-resolution structure of the full assembly absent","How substrate engagement competes with 14-3-3 not directly visualized"]},{"year":null,"claim":"Whether the diverse single-study substrate and ferroptosis-related claims reflect a unified mechanism or context-specific regulation, and which substrates dominate each physiological setting, remains unresolved.","evidence":"Open question across the corpus","pmids":[],"confidence":"Low","gaps":["Many ferroptosis substrates (GPX4, ACSL4, STAT3, ENO1, YBX1) rest on Low-confidence single Co-IPs","In vivo substrate hierarchy outside kidney/lung not established"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0140096","term_label":"catalytic activity, acting on a protein","supporting_discovery_ids":[6,11,14,17,26,32]},{"term_id":"GO:0016874","term_label":"ligase activity","supporting_discovery_ids":[6,7,17,32]},{"term_id":"GO:0098772","term_label":"molecular function regulator activity","supporting_discovery_ids":[0,4,8]}],"localization":[{"term_id":"GO:0005886","term_label":"plasma membrane","supporting_discovery_ids":[6,11,20]},{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[17,24]}],"pathway":[{"term_id":"R-HSA-382551","term_label":"Transport of small molecules","supporting_discovery_ids":[6,11,12,20]},{"term_id":"R-HSA-392499","term_label":"Metabolism of proteins","supporting_discovery_ids":[6,7,17,32]},{"term_id":"R-HSA-168256","term_label":"Immune System","supporting_discovery_ids":[14,18,32,33]},{"term_id":"R-HSA-162582","term_label":"Signal Transduction","supporting_discovery_ids":[17,22,24,49]},{"term_id":"R-HSA-112316","term_label":"Neuronal System","supporting_discovery_ids":[13,29,40]}],"complexes":[],"partners":["SGK1","YWHAB","SCNN1A","SLC12A3","GRK2","NDFIP1","AMOTL1","USP7"],"other_free_text":[]}},"prefetch_data":{"uniprot":{"accession":"Q96PU5","full_name":"E3 ubiquitin-protein ligase NEDD4-like","aliases":["HECT-type E3 ubiquitin transferase NED4L","NEDD4.2","Nedd4-2"],"length_aa":975,"mass_kda":111.9,"function":"E3 ubiquitin-protein ligase that mediates the polyubiquitination of lysine and cysteine residues on target proteins and is thereby implicated in the regulation of various signaling pathways including autophagy, innate immunity or DNA repair (PubMed:20064473, PubMed:31959741, PubMed:33608556). Inhibits TGF-beta signaling by triggering SMAD2 and TGFBR1 ubiquitination and proteasome-dependent degradation (PubMed:15496141). Downregulates autophagy and cell growth by ubiquitinating and reducing cellular ULK1 or ASCT2 levels (PubMed:28820317, PubMed:31959741). Promotes ubiquitination and internalization of various plasma membrane channels such as ENaC, SCN2A/Nav1.2, SCN3A/Nav1.3, SCN5A/Nav1.5, SCN9A/Nav1.7, SCN10A/Nav1.8, KCNA3/Kv1.3, KCNH2, EAAT1, KCNQ2/Kv7.2, KCNQ3/Kv7.3 or CLC5 (PubMed:26363003, PubMed:27445338). Promotes ubiquitination and degradation of SGK1 and TNK2. Ubiquitinates BRAT1 and this ubiquitination is enhanced in the presence of NDFIP1 (PubMed:25631046). Plays a role in dendrite formation by melanocytes (PubMed:23999003). Involved in the regulation of TOR signaling (PubMed:27694961). Ubiquitinates and regulates protein levels of NTRK1 once this one is activated by NGF (PubMed:27445338). Plays a role in antiviral innate immunity by catalyzing 'Lys-29'-linked cysteine ubiquitination of TRAF3, resulting in enhanced 'Lys-48' and 'Lys-63'-linked ubiquitination of TRAF3 (PubMed:33608556). Ubiquitinates TTYH2 and TTYH3 and regulates protein levels of TTYH2 (PubMed:18577513)","subcellular_location":"Cytoplasm; Golgi apparatus; Endosome, multivesicular body","url":"https://www.uniprot.org/uniprotkb/Q96PU5/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/NEDD4L","classification":"Not Classified","n_dependent_lines":5,"n_total_lines":1208,"dependency_fraction":0.0041390728476821195},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[],"url":"https://opencell.sf.czbiohub.org/search/NEDD4L","total_profiled":1310},"omim":[{"mim_id":"621529","title":"TRANSMEMBRANE PROTEIN 168; TMEM168","url":"https://www.omim.org/entry/621529"},{"mim_id":"617986","title":"LOW DENSITY LIPOPROTEIN RECEPTOR CLASS A DOMAIN-CONTAINING PROTEIN 3; LDLRAD3","url":"https://www.omim.org/entry/617986"},{"mim_id":"617476","title":"CNKSR FAMILY, MEMBER 3; CNKSR3","url":"https://www.omim.org/entry/617476"},{"mim_id":"617201","title":"PERIVENTRICULAR NODULAR HETEROTOPIA 7; PVNH7","url":"https://www.omim.org/entry/617201"},{"mim_id":"614436","title":"CHARCOT-MARIE-TOOTH DISEASE, AXONAL, TYPE 2P; CMT2P","url":"https://www.omim.org/entry/614436"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Low tissue specificity","tissue_distribution":"Detected in all","driving_tissues":[],"url":"https://www.proteinatlas.org/search/NEDD4L"},"hgnc":{"alias_symbol":["KIAA0439","RSP5","NEDD4-2"],"prev_symbol":[]},"alphafold":{"accession":"Q96PU5","domains":[{"cath_id":"2.60.40.150","chopping":"8-151","consensus_level":"high","plddt":87.8653,"start":8,"end":151},{"cath_id":"2.20.70.10","chopping":"553-582","consensus_level":"high","plddt":77.64,"start":553,"end":582},{"cath_id":"3.30.2160.10","chopping":"660-852","consensus_level":"high","plddt":89.7149,"start":660,"end":852},{"cath_id":"3.30.2410.10","chopping":"859-966","consensus_level":"high","plddt":87.5642,"start":859,"end":966}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96PU5","model_url":"https://alphafold.ebi.ac.uk/files/AF-Q96PU5-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-Q96PU5-F1-predicted_aligned_error_v6.png","plddt_mean":68.38},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=NEDD4L","jax_strain_url":"https://www.jax.org/strain/search?query=NEDD4L"},"sequence":{"accession":"Q96PU5","fasta_url":"https://rest.uniprot.org/uniprotkb/Q96PU5.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/Q96PU5/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/Q96PU5"}},"corpus_meta":[{"pmid":"11742982","id":"PMC_11742982","title":"Phosphorylation of Nedd4-2 by Sgk1 regulates epithelial Na(+) channel cell surface expression.","date":"2001","source":"The EMBO journal","url":"https://pubmed.ncbi.nlm.nih.gov/11742982","citation_count":570,"is_preprint":false},{"pmid":"11696533","id":"PMC_11696533","title":"Serum and glucocorticoid-regulated kinase modulates Nedd4-2-mediated inhibition of the epithelial Na+ channel.","date":"2001","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/11696533","citation_count":357,"is_preprint":false},{"pmid":"19557014","id":"PMC_19557014","title":"Nedd4 and Nedd4-2: closely related ubiquitin-protein ligases with distinct physiological functions.","date":"2010","source":"Cell death and differentiation","url":"https://pubmed.ncbi.nlm.nih.gov/19557014","citation_count":194,"is_preprint":false},{"pmid":"9108033","id":"PMC_9108033","title":"The large subunit of RNA polymerase II is a substrate of the Rsp5 ubiquitin-protein ligase.","date":"1997","source":"Proceedings of the National Academy of Sciences of the United States of America","url":"https://pubmed.ncbi.nlm.nih.gov/9108033","citation_count":186,"is_preprint":false},{"pmid":"17502380","id":"PMC_17502380","title":"Nedd4-2 catalyzes ubiquitination and degradation of cell surface ENaC.","date":"2007","source":"The Journal of biological chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/17502380","citation_count":179,"is_preprint":false},{"pmid":"25344756","id":"PMC_25344756","title":"Rsp5/Nedd4 is the main ubiquitin ligase that targets cytosolic misfolded proteins following heat stress.","date":"2014","source":"Nature cell biology","url":"https://pubmed.ncbi.nlm.nih.gov/25344756","citation_count":157,"is_preprint":false},{"pmid":"12167587","id":"PMC_12167587","title":"Concerted action of ENaC, Nedd4-2, and Sgk1 in transepithelial Na(+) transport.","date":"2002","source":"American journal of physiology. 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a recombinant dominant-negative WW domain construct interferes with Na+-dependent feedback inhibition of ENaC in whole-cell patch-clamp experiments.\",\n      \"method\": \"GST pulldown, whole-cell patch-clamp electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct binding and functional electrophysiology in single study\",\n      \"pmids\": [\"11244092\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2002,\n      \"finding\": \"Only WW3 and WW4 domains of Nedd4-2 are required for both binding to ENaC subunits and regulation of Na+ feedback control; both domains together are essential for in vivo function, indicating they interact with distinct non-interchangeable sites in ENaC.\",\n      \"method\": \"Surface plasmon resonance binding kinetics, whole-cell patch-clamp in mammalian cells, Xenopus oocyte expression\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — two orthogonal methods (binding kinetics + electrophysiology) in single laboratory\",\n      \"pmids\": [\"12424229\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"14-3-3 proteins bind directly to SGK1-phosphorylated Nedd4-2 at Ser-468, preserving the activity of SGK1-stimulated ENaC-dependent Na+ current; disrupting the 14-3-3/Nedd4-2 interaction decreases ENaC surface density, likely by allowing Nedd4-2-mediated ubiquitination and ENaC degradation.\",\n      \"method\": \"Co-immunoprecipitation, Xenopus oocyte electrophysiology, surface biotinylation\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — direct interaction mapping with phosphosite, functional readout, replicated with structural detail in PMID:16716084 and PMID:34294877\",\n      \"pmids\": [\"15677482\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"14-3-3 proteins inhibit the interaction between WW domains of hNedd4-2 and the PY motif of ENaC in a manner dependent on SGK1-catalyzed phosphorylation of Ser468 (and similarly PKA-mediated phosphorylation), shielding phosphorylated Nedd4-2 from dephosphorylation.\",\n      \"method\": \"Surface plasmon resonance spectrometry, Xenopus oocyte electrophysiology\",\n      \"journal\": \"Biochemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — surface plasmon resonance quantitative binding assay plus functional oocyte data, consistent with prior study\",\n      \"pmids\": [\"16716084\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"Nedd4-2 catalyzes ubiquitination of α-, β-, and γENaC subunits at the cell surface; monoubiquitination is sufficient to reduce ENaC surface expression and current; HECT domain mutation abolishes activity; Nedd4-2 selectively reduces surface ENaC without affecting immature biosynthetic ENaC.\",\n      \"method\": \"Ubiquitination assays, surface biotinylation, siRNA knockdown, overexpression, electrophysiology\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (ubiquitination assay, biotinylation, electrophysiology, siRNA/overexpression) in rigorous single study\",\n      \"pmids\": [\"17502380\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2008,\n      \"finding\": \"The WW domains of Nedd4-2 bind an intramolecular PY motif (LPXY) within its own HECT domain, inhibiting auto-ubiquitination and stabilizing Nedd4-2; mutation of this HECT PY motif decreases Nedd4-2 stability without affecting ENaC ubiquitination; ENaC binding disrupts this intramolecular interaction, triggering Nedd4-2 self-ubiquitination and degradation.\",\n      \"method\": \"Pulse-chase experiments, co-immunoprecipitation, mutational analysis, ubiquitination assays\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (pulse-chase, Co-IP, mutagenesis, ubiquitination assay) in single study revealing a novel autoinhibitory mechanism\",\n      \"pmids\": [\"18498246\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2004,\n      \"finding\": \"GRK2 phosphorylates the C terminus of the ENaC β subunit and renders ENaC insensitive to inhibition by Nedd4-2, maintaining channels in the active state; this represents a mechanism by which GRK2 counteracts Nedd4-2-dependent channel inhibition.\",\n      \"method\": \"Xenopus oocyte electrophysiology, in vitro kinase assay, epistasis experiments\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — in vitro kinase assay plus functional oocyte electrophysiology in single study\",\n      \"pmids\": [\"15284439\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2007,\n      \"finding\": \"GRK2 physically interacts with both Nedd4 and Nedd4-2, and phosphorylates both ligases at multiple sites including Thr466 in the WW3 domain region of Nedd4.\",\n      \"method\": \"Co-immunoprecipitation, in vitro phosphorylation assay\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP and in vitro kinase assay in single study, extends mechanistic understanding from PMID:15284439\",\n      \"pmids\": [\"17544362\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Knockout of Nedd4-2 in mice leads to increased ENaC expression and activity in embryonic lung, causing premature fetal lung fluid clearance and perinatal lethality; surviving knockouts develop lethal sterile lung inflammation, establishing Nedd4-2 as an essential regulator of ENaC-dependent lung fluid clearance in vivo.\",\n      \"method\": \"Genetic knockout mouse model, electrophysiology, immunoblotting\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — clean in vivo knockout with defined molecular and physiological phenotype, multiple orthogonal readouts\",\n      \"pmids\": [\"21505443\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2011,\n      \"finding\": \"Nedd4-2 co-immunoprecipitates with NCC and stimulates NCC ubiquitylation at the cell surface; catalytically inactive Nedd4-2 fails to decrease NCC activity; SGK1 prevents this inhibition in a kinase-dependent manner; double phosphorylation of Nedd4-2 at Ser222/Ser328 abolishes SGK1-mediated rescue. Unlike ENaC regulation, this does not require a PY motif in NCC.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, Xenopus oocyte electrophysiology, surface biotinylation, knockout mice\",\n      \"journal\": \"Journal of the American Society of Nephrology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods (Co-IP, ubiquitination, electrophysiology, KO mice) in single rigorous study\",\n      \"pmids\": [\"21852580\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2013,\n      \"finding\": \"Inducible, nephron-specific Nedd4-2 knockout in mice causes salt-sensitive hypertension characterized by increased NCC, β/γENaC (but not αENaC) and ROMK expression; thiazide treatment reverses hypertension, demonstrating NCC as a primary in vivo target of NEDD4-2 in the kidney.\",\n      \"method\": \"Conditional knockout mouse model, immunoblotting, blood pressure telemetry, pharmacological rescue\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — inducible cell-type-specific KO with defined molecular phenotype and pharmacological rescue, multiple independent readouts\",\n      \"pmids\": [\"23348737\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2014,\n      \"finding\": \"Nedd4-2 regulates voltage-gated sodium channel (Nav) activity specifically in response to elevated intracellular Na+ in fetal cortical neurons but does not affect steady-state Nav activity, as shown using Nedd4-2-deficient mice; no such regulation was observed in dorsal root ganglia neurons from the same mice.\",\n      \"method\": \"Nedd4-2 knockout mouse neurons, electrophysiology, intracellular Na+ manipulation\",\n      \"journal\": \"The Biochemical journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — clean KO model with defined electrophysiological phenotype in specific cell type, single laboratory\",\n      \"pmids\": [\"24152020\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NEDD4-2 ubiquitinates TRAF3 at Cys56 and Cys124 (zinc finger residues) via K29-linked cysteine ubiquitination; this promotes enhanced TRAF3 association with K48/K63 E3 ligases (cIAP1/2, HECTD3) and TRAF3-TBK1 interaction, leading to TBK1 and IRF3 phosphorylation and type I interferon production upon virus infection.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mass spectrometry, site-directed mutagenesis, knockout cells and mice\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including MS, mutagenesis, Co-IP, KO in vitro and in vivo in single rigorous study\",\n      \"pmids\": [\"33608556\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nedd4-2 ubiquitinates and downregulates the cardiac potassium channel hERG1 via a PY motif in the C-terminus of hERG1; Nedd4-2 co-expression decreases total and surface hERG1 protein and whole-cell current; deletion of the PY motif abrogates Nedd4-2-dependent regulation.\",\n      \"method\": \"Co-immunoprecipitation, surface biotinylation, Western blot, electrophysiology in HEK293 cells\",\n      \"journal\": \"Journal of molecular and cellular cardiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — reciprocal Co-IP, biotinylation, and electrophysiology in single lab study\",\n      \"pmids\": [\"21463633\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nedd4-2 ubiquitinates and decreases surface expression and activity of organic anion transporter OAT3; this requires the WW3 and WW4 domains of Nedd4-2; a physical interaction between OAT3 and Nedd4-2 was confirmed in cultured cells and rat kidney slices.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, transport activity assay, siRNA knockdown, mutagenesis\",\n      \"journal\": \"Molecular pharmaceutics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (Co-IP, ubiquitination assay, transport assay, mutagenesis) in single laboratory\",\n      \"pmids\": [\"26651153\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NEDD4L binds to and promotes ubiquitination and proteasome-dependent degradation of PIK3CA (p110α catalytic subunit of PI3K), negatively regulating PIK3CA protein levels and affecting PI3K-AKT signaling; NEDD4L does not ubiquitinate the PI3K regulatory subunit.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cycloheximide chase, overexpression/knockdown with immunoblotting\",\n      \"journal\": \"The Journal of biological chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple biochemical methods in single laboratory\",\n      \"pmids\": [\"27339899\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nedd4-2 ubiquitinates phosphorylated Syk (a tyrosine kinase essential for FcɛRI signaling) with adaptor protein Ndfip1, limiting the intensity and duration of IgE-FcɛRI signaling in mast cells; loss of Nedd4-2 or Ndfip1 in mast cells causes exacerbated cutaneous anaphylaxis in vivo.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mast cell-specific knockout mice, in vivo anaphylaxis model\",\n      \"journal\": \"Nature communications\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — cell-type-specific KO with in vivo phenotype, biochemical validation of substrate ubiquitination, single rigorous study\",\n      \"pmids\": [\"27786273\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"GPCR PAR1 stimulates c-Src-mediated tyrosine phosphorylation of NEDD4-2 at Y485 (within the linker between WW2 and WW3 domains), activating NEDD4-2 E3 ligase activity and driving p38 signaling and endothelial barrier disruption; Y485 mutation impairs E3 ligase activity and p38 activation.\",\n      \"method\": \"Mass spectrometry phosphoproteomics, site-directed mutagenesis, kinase inhibitor experiments, endothelial permeability assays\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1-2 / Moderate — MS phosphosite identification, mutagenesis, functional rescue in single rigorous study\",\n      \"pmids\": [\"30232011\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2018,\n      \"finding\": \"Nedd4-2 binds to Kir5.1 via a phosphothreonine (TPVT) motif at its C-terminus; this interaction facilitates ubiquitination of the Kir4.1/Kir5.1 heterotetramer (not Kir4.1 alone) and reduces Kir4.1 expression and K+ currents in the distal convoluted tubule.\",\n      \"method\": \"Co-immunoprecipitation, GST pulldown, ubiquitination assay, patch-clamp electrophysiology, Kir5.1 mutagenesis, kidney-specific Nedd4-2 knockout mice\",\n      \"journal\": \"American journal of physiology. Renal physiology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods including KO mice, mutagenesis, electrophysiology, pulldown and ubiquitination assays\",\n      \"pmids\": [\"29897283\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NEDD4L binds ULK1 in pancreatic cancer cells and promotes its ubiquitination and degradation; NEDD4L depletion stabilizes ULK1, enhancing autophagy activity and mitochondrial metabolic function; NEDD4L also regulates the glutamine transporter ASCT2.\",\n      \"method\": \"Co-immunoprecipitation, siRNA knockdown, immunoblotting, oxygen consumption rate measurement, xenograft mouse models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, knockdown with functional readout, in vivo xenograft, single laboratory\",\n      \"pmids\": [\"31959741\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"NEDD4 and NEDD4L negatively regulate Wnt/β-catenin signaling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation; loss of both ligases enhances ISC proliferation, increases RSPO sensitivity, and accelerates tumor development in Apcmin mice.\",\n      \"method\": \"Conditional knockout mice, co-immunoprecipitation, ubiquitination assay, organoid culture, tumor phenotyping\",\n      \"journal\": \"The EMBO journal\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP and KO with defined phenotype, but involves both NEDD4 and NEDD4L making NEDD4L-specific contribution partially confounded\",\n      \"pmids\": [\"31867777\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4L identifies and promotes ubiquitination and proteasomal degradation of lactotransferrin (LTF) via direct protein-protein interaction, thereby inhibiting intracellular iron accumulation and ferroptotic cell death.\",\n      \"method\": \"siRNA screen, mass spectrometry for substrate identification, co-immunoprecipitation, ubiquitination assay, ferroptosis cell death assays\",\n      \"journal\": \"Biochemical and biophysical research communications\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — MS-based substrate identification, Co-IP, functional ferroptosis assay in single laboratory\",\n      \"pmids\": [\"32811647\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4L ubiquitinates and promotes degradation of SMAD2, a TGFβ pathway transcription modulator; USP7 deubiquitinates NEDD4L (stabilizing it), and this USP7-NEDD4L-SMAD2 axis controls autophagy and protein quality control in a neuroprotective pathway conserved from C. elegans to mammals.\",\n      \"method\": \"Genetic epistasis in C. elegans and Drosophila, co-immunoprecipitation, ubiquitination assay in mammalian cells\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — cross-species epistasis plus biochemical validation, but mechanistic detail of NEDD4L-SMAD2 interaction assessed in single lab\",\n      \"pmids\": [\"33106424\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4-2 ubiquitinates α-synuclein after focal ischemia; Nedd4l knockout reduces post-ischemic poly-ubiquitinated α-synuclein and exacerbates ischemic brain damage; co-immunoprecipitation confirms enhanced α-synuclein–Nedd4l interaction after stroke.\",\n      \"method\": \"Transient MCAO mouse model, knockout mice, co-immunoprecipitation, behavioral testing\",\n      \"journal\": \"Journal of cerebral blood flow and metabolism\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — KO mouse with defined phenotype, Co-IP for interaction, single laboratory\",\n      \"pmids\": [\"32703111\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4-2 mediates ubiquitination and degradation of Merlin/NF2 (mostly mono/di-ubiquitination at K396); this requires the scaffold protein AMOTL1 to bridge NEDD4L to Merlin; ubiquitination is promoted by Ser518 dephosphorylation in response to Ca2+ signaling or cell detachment, and is required for Merlin binding to Lats1 and Hippo pathway activation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mutagenesis, Ca2+ signaling experiments, Lats1 kinase activity assay\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods (Co-IP, ubiquitination, mutagenesis, kinase assay) in single laboratory\",\n      \"pmids\": [\"33058421\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nedd4-2 ubiquitinates the SP-C proprotein at K6 (via its PY motif binding to Nedd4-2 WW domains), and this ubiquitination is required for lumenal relocation of SP-C within multivesicular bodies, leading to SP-C processing and secretion.\",\n      \"method\": \"GST pulldown with recombinant proteins, co-immunoprecipitation, ubiquitination assay, alanine mutagenesis, adenoviral infection in type II cells\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (pulldown, Co-IP, ubiquitination, mutagenesis, functional secretion assay) in single laboratory\",\n      \"pmids\": [\"19423771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"NDFIP2 (and NDFIP1) serve as adaptors that bridge NEDD4/NEDD4L to AQP2, enabling NEDD4/NEDD4L-mediated ubiquitination and degradation of AQP2; NEDD4 and NEDD4L do not bind AQP2 directly.\",\n      \"method\": \"Membrane Yeast Two-Hybrid, siRNA knockdown, co-immunoprecipitation, ubiquitination assay, immunoblotting in HEK293 and mpkCCD cells\",\n      \"journal\": \"PloS one\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple methods identifying indirect substrate engagement mechanism; single laboratory\",\n      \"pmids\": [\"28931009\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2017,\n      \"finding\": \"Nedd4-2 ubiquitinates epilepsy-associated GluA1 subunit of AMPA receptors, reducing GluA1 surface expression; epilepsy-associated missense mutations in Nedd4-2 disrupt GluA1 ubiquitination; genetic reduction of GluA1 normalizes seizure susceptibility in Nedd4-2andi mice.\",\n      \"method\": \"Knockout/knock-in mouse model, ubiquitination assay, surface biotinylation, extracellular electrophysiology, in vivo seizure model, genetic epistasis\",\n      \"journal\": \"PLoS genetics\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple orthogonal methods plus in vivo genetic epistasis rescue, rigorous mechanistic study\",\n      \"pmids\": [\"28212375\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"14-3-3 protein binding to phosphorylated Ser342 and Ser448 of Nedd4-2 induces a structural rearrangement that inhibits intramolecular interactions between the HECT and WW domains, providing the structural basis for 14-3-3-mediated Nedd4-2 regulation.\",\n      \"method\": \"Integrative structural biology (SAXS, NMR, HDX-MS), mutagenesis, phosphopeptide binding assays\",\n      \"journal\": \"Communications biology\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — integrative structural biology plus functional validation, multiple orthogonal biophysical methods in single study\",\n      \"pmids\": [\"34294877\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2019,\n      \"finding\": \"PLPP/CIN (pyridoxal-5'-phosphate phosphatase/chronophin) specifically dephosphorylates NEDD4-2 at Ser448, reducing Nedd4-2 ubiquitination and facilitating NEDD4-2-mediated ubiquitination of GluA1, thereby modulating neuronal excitability.\",\n      \"method\": \"In vitro phosphatase assay, co-immunoprecipitation, ubiquitination assay, electrophysiology, knockout cell models\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — direct phosphatase assay plus functional ubiquitination and electrophysiology in single laboratory\",\n      \"pmids\": [\"31320629\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L directly interacts with MEKK2 and promotes its poly-ubiquitination and degradation; IL-17-induced Ser520 phosphorylation of MEKK2 is required for NEDD4L-mediated MEKK2 degradation and shutdown of IL-17R signaling; Nedd4l-deficient mice show increased susceptibility to IL-17-induced inflammation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mutagenesis, knockout mice, experimental autoimmune encephalomyelitis (EAE) model\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"High\",\n      \"confidence_rationale\": \"Tier 2 / Strong — multiple methods (Co-IP, ubiquitination, mutagenesis, KO mice with in vivo disease model), rigorous single-laboratory study\",\n      \"pmids\": [\"36161689\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L directly interacts with GP130 and mediates its Lys-27-linked ubiquitination and proteasomal degradation, negatively regulating IL-6/GP130/STAT3 signaling in keratinocytes and suppressing skin hyperplasia.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mutagenesis, knockout mice (imiquimod skin model), immunohistochemistry\",\n      \"journal\": \"EMBO reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, and KO mice with phenotypic readout in single laboratory\",\n      \"pmids\": [\"33769697\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L directly interacts with GPX4 and promotes its ubiquitination and degradation, facilitating ferroptosis in granulosa cells.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, cell viability assay, ferroptosis marker measurement\",\n      \"journal\": \"Endocrine connections\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and ubiquitination assay, single laboratory, limited mechanistic follow-up\",\n      \"pmids\": [\"36662677\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L promotes ubiquitination and degradation of ENO1 (enolase 1), suppressing glycolysis and proliferation in oral squamous cell carcinoma; ENO1 overexpression reverses functional effects of NEDD4L overexpression.\",\n      \"method\": \"Ubiquitination assay, co-immunoprecipitation, overexpression/knockdown with glycolysis measurements, in vivo tumor model\",\n      \"journal\": \"Cancer biology & therapy\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — computational substrate prediction followed by single Co-IP and ubiquitination assay in single laboratory\",\n      \"pmids\": [\"35316145\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L mediates ubiquitination and degradation of YBX1 protein when recruited by circNEIL3; direct interaction between NEDD4L and YBX1 was confirmed, and this promotes proteasomal degradation of YBX1 to suppress tumor metastasis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, loss-of-function/overexpression experiments in cancer cells and in vivo\",\n      \"journal\": \"Proceedings of the National Academy of Sciences of the United States of America\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — mechanistic finding about NEDD4L-YBX1 interaction is secondary to the circRNA study; single lab, single Co-IP\",\n      \"pmids\": [\"36961927\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L ubiquitinates and degrades ACSL4, a key ferroptosis regulator; YAP facilitates NEDD4L transcription, so YAP→NEDD4L→ACSL4 ubiquitination axis reduces ferroptosis in myocardial ischemia-reperfusion injury.\",\n      \"method\": \"Co-immunoprecipitation, ChIP, dual-luciferase reporter assay, Western blot, in vivo MIRI model\",\n      \"journal\": \"The Canadian journal of cardiology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for NEDD4L-ACSL4 interaction, transcriptional mechanism for YAP-NEDD4L included, single laboratory\",\n      \"pmids\": [\"37541340\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"14-3-3σ promotes binding of S448-phosphorylated NEDD4L to HIF-1α, enhancing HIF-1α poly-ubiquitination and proteasomal degradation; this suppresses hypoxia-induced tumor progression in colorectal cancer.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mutagenesis, patient sample correlation analysis\",\n      \"journal\": \"Cell reports\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, mutagenesis in single laboratory with clinical correlation\",\n      \"pmids\": [\"37494179\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2024,\n      \"finding\": \"NEDD4L ubiquitinates and degrades TFRC (transferrin receptor/CD71), reducing iron accumulation; METTL3-mediated m6A methylation stabilizes NEDD4L mRNA, enhancing NEDD4L expression and the NEDD4L-TFRC axis to inhibit ferroptosis and neuronal damage in ischemic stroke.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, half-life experiments, MCAO mouse model, NEDD4L knockout model\",\n      \"journal\": \"Cell biology and toxicology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, KO model, in vivo MCAO, multiple methods in single laboratory\",\n      \"pmids\": [\"38302612\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2021,\n      \"finding\": \"Non-SUMOylated CRMP2 forms a complex with Numb, Eps15, and Nedd4-2 to promote clathrin-mediated endocytosis of NaV1.7; silencing Nedd4-2 in DRG neurons from CRMP2K374A/K374A mice restores NaV1.7 surface expression and sodium currents.\",\n      \"method\": \"siRNA knockdown in DRG neurons, electrophysiology, in vivo nerve injury model\",\n      \"journal\": \"Molecular brain\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — genetic approach in primary neurons with electrophysiological readout; NEDD4L role inferred from complex membership and siRNA data\",\n      \"pmids\": [\"33478555\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2020,\n      \"finding\": \"NEDD4L interacts with Piezo2 in co-immunoprecipitation experiments in HEK293T cells and inhibits Piezo2 mechanically-activated currents when co-expressed; Nedd4-2 upregulation in hypertensive rat nodose ganglia correlates with Piezo2 downregulation.\",\n      \"method\": \"Co-immunoprecipitation, electrophysiology in HEK293T, in vivo knockdown, spontaneously hypertensive rat model\",\n      \"journal\": \"Pharmacological research\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and electrophysiology, correlational in vivo data, single laboratory\",\n      \"pmids\": [\"33352230\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L deficiency in intestinal epithelial cells inhibits expression of SLC3A2 (protein level only, not mRNA), reducing GPX4 expression and promoting ferroptosis; this mechanistic axis (NEDD4L→SLC3A2→GPX4) maintains intestinal homeostasis and restricts inflammatory bowel disease.\",\n      \"method\": \"Conditional and global KO mice, DSS/TNBS colitis and AOM-DSS tumor models, immunoblotting, ferroptosis inhibitor rescue\",\n      \"journal\": \"The Journal of clinical investigation\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple KO models with defined phenotype and pharmacological rescue; mechanistic link to SLC3A2 post-translational regulation is partially indirect\",\n      \"pmids\": [\"39688910\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2005,\n      \"finding\": \"Both Nedd4 and Nedd4-2 use UbcH5b as their primary E2 ubiquitin-conjugating enzyme in in vitro ubiquitination assays; Ube2e3 is used less efficiently by both.\",\n      \"method\": \"In vitro ubiquitination assay with panel of E2 enzymes\",\n      \"journal\": \"The international journal of biochemistry & cell biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 1 / Moderate — reconstituted in vitro ubiquitination assay; single laboratory but clean biochemical result\",\n      \"pmids\": [\"16337426\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2009,\n      \"finding\": \"NEDD4L directly binds surfactant protein-C (SP-C) proprotein via interaction of its WW domain with the PPDY motif in SP-C residues 12-17; NEDD4L ubiquitinates SP-C at K6 (but not K34); this ubiquitination is required for SP-C proprotein processing.\",\n      \"method\": \"GST pulldown with recombinant proteins, endogenous Co-IP from lung lysates, ubiquitination assay, mutagenesis, SP-C secretion assay\",\n      \"journal\": \"American journal of respiratory cell and molecular biology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple orthogonal methods (GST pulldown, endogenous Co-IP, ubiquitination assay, mutagenesis, functional secretion assay) in single laboratory\",\n      \"pmids\": [\"19423771\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2016,\n      \"finding\": \"NEDD4L overexpression up-regulates autophagy; knockdown suppresses both basal and ER stress-induced autophagy in cultured cells; Nedd4-2 expression is induced by ER stress via IRE1/XBP1s pathway, linking UPR to autophagic response.\",\n      \"method\": \"Pharmacological ER stress induction, siRNA knockdown, overexpression, IRE1 inhibition, immunoblotting for autophagy markers\",\n      \"journal\": \"FASEB journal\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — functional cell biology without direct substrate identification for autophagy regulation; single laboratory\",\n      \"pmids\": [\"27022162\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"Decreased NEDD4L expression leads to impaired TXNIP ubiquitination and its accumulation in NASH liver; NEDD4L-mediated TXNIP degradation is part of a NEDD4L-TXNIP-CHOP axis that controls hepatocyte apoptosis in nonalcoholic steatohepatitis.\",\n      \"method\": \"Four independent NASH mouse models, gain/loss-of-function studies, co-immunoprecipitation, ubiquitination assay\",\n      \"journal\": \"Theranostics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — multiple animal models, Co-IP, ubiquitination assay; NEDD4L-TXNIP interaction mechanistically validated\",\n      \"pmids\": [\"37153733\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L mediates ubiquitination and degradation of STAT3, as shown by co-immunoprecipitation in glioma cells; NEDD4L-induced ferroptosis is rescued by forced expression of STAT3, placing STAT3 downstream of NEDD4L in glioma ferroptosis regulation.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, genetic rescue experiments, xenograft mouse model\",\n      \"journal\": \"Journal of oncology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP and rescue experiment in single laboratory\",\n      \"pmids\": [\"36618071\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"NEDD4L binds the 19S proteasome and promotes autophagy; NEDD4L suppression reduces bortezomib sensitivity in multiple myeloma through autophagy inhibition.\",\n      \"method\": \"Co-immunoprecipitation with proteasome components, knockdown with autophagy and viability assays, in vivo mouse model\",\n      \"journal\": \"Cell death & disease\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 / Weak — single Co-IP for proteasome binding, functional autophagy data without defined substrate; single laboratory\",\n      \"pmids\": [\"35236820\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L ubiquitinates and degrades TβRII (TGF-β receptor II), the first signaling enzyme of TGF-β1 pathway, suppressing lung myofibroblast differentiation; transcription factor E2F inhibits NEDD4L expression, and blocking E2F rescues NEDD4L levels and mitigates experimental pulmonary fibrosis.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, knockdown/overexpression, E2F inhibitor experiments, animal model of pulmonary fibrosis\",\n      \"journal\": \"Translational research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination assay, in vivo model; single laboratory\",\n      \"pmids\": [\"36257596\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"NEDD4L mediates ubiquitination and degradation of ACE2 in neurogenic hypertension; Ang-II-induced upregulation of Nedd4-2 in the brain promotes ACE2 ubiquitination; mutation of lysine residues in the ACE2 C-terminus confers ubiquitination resistance.\",\n      \"method\": \"Co-immunoprecipitation, ubiquitination assay, mutagenesis, gene therapy in brain regions, blood pressure telemetry\",\n      \"journal\": \"Cardiovascular research\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 / Moderate — Co-IP, ubiquitination, mutagenesis, in vivo optogenetics/telemetry; single laboratory\",\n      \"pmids\": [\"37161607\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"NEDD4L (NEDD4-2) is a HECT-type E3 ubiquitin ligase whose WW domains bind PPxY (PY) motifs in substrate proteins to catalyze their ubiquitination, targeting them for endocytosis and/or proteasomal/lysosomal degradation; its best-characterized substrates include the epithelial Na+ channel ENaC, the Na+-Cl- cotransporter NCC, voltage-gated Na+ and K+ channels, AMPA receptor GluA1, and numerous signaling proteins (TRAF3, MEKK2, PIK3CA, LGR5/DVL2, Merlin/NF2, SMAD2, HIF-1α, TβRII, ACE2, and others); its activity is tightly regulated by SGK1-mediated phosphorylation (which recruits 14-3-3 proteins to sterically inhibit WW-PY interactions), by c-Src-mediated tyrosine phosphorylation at Y485 (which activates it downstream of GPCRs), by PLPP/CIN-mediated dephosphorylation, and by an intramolecular autoinhibitory interaction between its own HECT-domain PY motif and its WW domains that is disrupted upon substrate engagement.\"\n}\n```","stage2_raw":"{\n  \"mechanistic_narrative\": \"NEDD4L (NEDD4-2) is a HECT-type E3 ubiquitin ligase that controls the surface abundance and activity of ion channels, transporters, receptors, and signaling proteins by catalyzing their ubiquitination, with UbcH5b serving as its primary E2 conjugating enzyme [#43]. Its WW domains engage PPxY (PY) motifs in substrates—paradigmatically all three ENaC subunits, where WW3 and WW4 contribute distinct non-interchangeable contacts—to drive substrate ubiquitination and endocytic/degradative removal from the cell surface [#2, #3, #6]. NEDD4L activity is gated by a multilayered phosphoregulatory network: SGK1 phosphorylates NEDD4-2 (at Ser444/Ser468 and related sites) to weaken WW-substrate engagement and recruit 14-3-3 proteins, which bind phospho-NEDD4-2 and impose a structural rearrangement that blocks intramolecular HECT-WW contacts and protects the phosphosites from dephosphorylation [#0, #1, #4, #5, #30]; conversely PLPP/CIN dephosphorylates Ser448 to license substrate ubiquitination [#31], and GPCR/c-Src signaling phosphorylates Tyr485 to activate the ligase [#19]. An autoinhibitory intramolecular interaction between the WW domains and a PY motif in NEDD4-2's own HECT domain stabilizes the ligase and is relieved upon substrate binding, coupling target engagement to self-ubiquitination [#7]. Through these mechanisms NEDD4L is an essential in vivo regulator of renal and pulmonary sodium handling—targeting ENaC, the Na-Cl cotransporter NCC, and Kir4.1/Kir5.1—such that its loss causes salt-sensitive hypertension and defective lung fluid clearance [#10, #11, #12, #20]. Beyond epithelial transport, NEDD4L ubiquitinates a broad substrate repertoire to shape neuronal excitability (GluA1, NaV channels) [#29, #13], innate and adaptive immune signaling (TRAF3, Syk, MEKK2, GP130) [#14, #18, #32, #33], and growth/stress pathways (PIK3CA, LGR5/DVL2, Merlin/NF2, SMAD2, TβRII, HIF-1α), often via adaptor- or scaffold-mediated substrate recruitment such as NDFIP1/2 and AMOTL1 [#17, #22, #26, #24, #49, #38, #28]. Epilepsy-associated missense mutations in NEDD4-2 disrupt GluA1 ubiquitination and alter seizure susceptibility, linking the ligase to neuronal disease [#29].\",\n  \"teleology\": [\n    {\n      \"year\": 2001,\n      \"claim\": \"Establishing how a kinase signal is transduced into ion-channel abundance, SGK1 was shown to phosphorylate NEDD4-2 via a PY-motif interaction, weakening NEDD4-2/ENaC binding and raising channel surface expression — defining the core SGK1-NEDD4-2-ENaC regulatory axis.\",\n      \"evidence\": \"Co-IP, phosphosite mapping, in vitro kinase assay, and electrophysiology in Xenopus oocytes\",\n      \"pmids\": [\"11742982\", \"11696533\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Did not resolve the structural basis of phospho-dependent WW disengagement\", \"Selectivity for hNedd4-2 over hNedd4 mechanistically unexplained at this stage\"]\n    },\n    {\n      \"year\": 2001,\n      \"claim\": \"To define substrate recognition, the WW domains of NEDD4L were shown to bind PPxY motifs in all three ENaC subunits, with a dominant-negative WW construct disrupting Na+ feedback inhibition — establishing direct WW-PY engagement as the recognition mode.\",\n      \"evidence\": \"GST pulldown and whole-cell patch-clamp\",\n      \"pmids\": [\"11244092\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Catalytic ubiquitination of ENaC not yet demonstrated\", \"Relative contribution of individual WW domains unresolved\"]\n    },\n    {\n      \"year\": 2002,\n      \"claim\": \"Refining the recognition map, WW3 and WW4 were shown to be the binding-competent domains contacting distinct non-interchangeable ENaC sites, both required for in vivo function.\",\n      \"evidence\": \"Surface plasmon resonance kinetics and patch-clamp\",\n      \"pmids\": [\"12424229\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Structural identity of the two distinct ENaC sites not defined\", \"Generality to non-ENaC substrates not tested\"]\n    },\n    {\n      \"year\": 2005,\n      \"claim\": \"Explaining how the SGK1 signal is sustained, 14-3-3 proteins were found to bind SGK1-phosphorylated NEDD4-2 at Ser468, preserving stimulated ENaC current — adding a second tier of regulation.\",\n      \"evidence\": \"Co-IP, oocyte electrophysiology, surface biotinylation\",\n      \"pmids\": [\"15677482\", \"16716084\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural mechanism of 14-3-3 inhibition not yet resolved\", \"Whether 14-3-3 acts on substrates beyond ENaC unknown\"]\n    },\n    {\n      \"year\": 2007,\n      \"claim\": \"Confirming catalytic action, NEDD4-2 was shown to ubiquitinate cell-surface ENaC subunits — monoubiquitination sufficing to reduce surface current — with HECT activity required, establishing ubiquitination as the effector step.\",\n      \"evidence\": \"Ubiquitination assays, biotinylation, siRNA/overexpression, electrophysiology\",\n      \"pmids\": [\"17502380\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Downstream endocytic/degradative machinery not defined\", \"Ubiquitin chain linkage on ENaC not specified\"]\n    },\n    {\n      \"year\": 2008,\n      \"claim\": \"Revealing intrinsic autoregulation, NEDD4-2 WW domains were found to bind an intramolecular HECT PY motif that suppresses auto-ubiquitination and stabilizes the ligase, with substrate binding disrupting this interaction — coupling target engagement to ligase turnover.\",\n      \"evidence\": \"Pulse-chase, Co-IP, mutagenesis, ubiquitination assays\",\n      \"pmids\": [\"18498246\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Structural conformation of the autoinhibited state not resolved here\", \"Interplay with 14-3-3 regulation not yet integrated\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Testing physiological essentiality, Nedd4-2 knockout mice revealed that the ligase is required in vivo for ENaC-dependent fetal lung fluid clearance, with loss causing perinatal lethality and lung inflammation.\",\n      \"evidence\": \"Genetic knockout mouse, electrophysiology, immunoblotting\",\n      \"pmids\": [\"21505443\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Tissue-specific contributions not dissected\", \"Adult renal phenotype not addressed in this model\"]\n    },\n    {\n      \"year\": 2011,\n      \"claim\": \"Extending substrate scope in the kidney, NEDD4-2 was shown to ubiquitinate NCC at the cell surface in a PY-motif-independent manner, with SGK1 antagonizing this regulation.\",\n      \"evidence\": \"Co-IP, ubiquitination assay, oocyte electrophysiology, biotinylation, KO mice\",\n      \"pmids\": [\"21852580\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Recognition mode for the PY-independent NCC interaction undefined\", \"Adaptor involvement not excluded\"]\n    },\n    {\n      \"year\": 2013,\n      \"claim\": \"Defining the dominant renal target, nephron-specific inducible Nedd4-2 knockout produced thiazide-reversible salt-sensitive hypertension with elevated NCC, establishing NCC as a primary in vivo kidney substrate.\",\n      \"evidence\": \"Conditional KO mouse, immunoblotting, blood pressure telemetry, pharmacological rescue\",\n      \"pmids\": [\"23348737\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Subunit-selective ENaC effects (β/γ but not α) mechanistically unexplained\", \"Relative quantitative contribution of NCC vs ENaC not parsed\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Broadening to channels, transporters, and signaling, NEDD4L was shown across studies to ubiquitinate Nav channels, hERG1, OAT3, Kir4.1/Kir5.1, GluA1, SP-C, AQP2, and to regulate immune and growth pathways via TRAF3, Syk, MEKK2, GP130, PIK3CA, LGR5/DVL2, Merlin/NF2, SMAD2, TβRII, HIF-1α and ferroptosis regulators.\",\n      \"evidence\": \"Co-IP, ubiquitination assays, mutagenesis, KO mice, and disease models across multiple substrates\",\n      \"pmids\": [\"24152020\", \"21463633\", \"26651153\", \"29897283\", \"28212375\", \"44\", \"28931009\", \"33608556\", \"27786273\", \"36161689\", \"33769697\", \"27339899\", \"31867777\", \"33058421\", \"33106424\", \"36257596\", \"37494179\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Many substrate interactions rest on single-laboratory Co-IP/ubiquitination data\", \"Ubiquitin chain linkages and adaptor requirements differ across substrates and are incompletely mapped\"]\n    },\n    {\n      \"year\": 2018,\n      \"claim\": \"Identifying an activating modification, GPCR-driven c-Src phosphorylation of NEDD4-2 at Tyr485 was shown to switch on E3 ligase activity and downstream p38 signaling — a regulatory input opposite to inhibitory phosphorylation.\",\n      \"evidence\": \"MS phosphoproteomics, mutagenesis, kinase inhibitors, endothelial permeability assays\",\n      \"pmids\": [\"30232011\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"Direct substrate of the activated ligase in this context not fully defined\", \"Structural effect of Y485 phosphorylation unresolved\"]\n    },\n    {\n      \"year\": 2021,\n      \"claim\": \"Providing the structural basis of inhibition, integrative structural biology showed 14-3-3 binding to phospho-Ser342/Ser448 rearranges NEDD4-2 to block intramolecular HECT-WW contacts, unifying the phospho/14-3-3 and autoinhibition models.\",\n      \"evidence\": \"SAXS, NMR, HDX-MS, mutagenesis, phosphopeptide binding\",\n      \"pmids\": [\"34294877\"],\n      \"confidence\": \"High\",\n      \"gaps\": [\"High-resolution structure of the full assembly absent\", \"How substrate engagement competes with 14-3-3 not directly visualized\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Whether the diverse single-study substrate and ferroptosis-related claims reflect a unified mechanism or context-specific regulation, and which substrates dominate each physiological setting, remains unresolved.\",\n      \"evidence\": \"Open question across the corpus\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"Many ferroptosis substrates (GPX4, ACSL4, STAT3, ENO1, YBX1) rest on Low-confidence single Co-IPs\", \"In vivo substrate hierarchy outside kidney/lung not established\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0140096\", \"supporting_discovery_ids\": [6, 11, 14, 17, 26, 32]},\n      {\"term_id\": \"GO:0016874\", \"supporting_discovery_ids\": [6, 7, 17, 32]},\n      {\"term_id\": \"GO:0140097\", \"supporting_discovery_ids\": []},\n      {\"term_id\": \"GO:0098772\", \"supporting_discovery_ids\": [0, 4, 8]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005886\", \"supporting_discovery_ids\": [6, 11, 20]},\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [17, 24]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-382551\", \"supporting_discovery_ids\": [6, 11, 12, 20]},\n      {\"term_id\": \"R-HSA-392499\", \"supporting_discovery_ids\": [6, 7, 17, 32]},\n      {\"term_id\": \"R-HSA-168256\", \"supporting_discovery_ids\": [14, 18, 32, 33]},\n      {\"term_id\": \"R-HSA-162582\", \"supporting_discovery_ids\": [17, 22, 24, 49]},\n      {\"term_id\": \"R-HSA-112316\", \"supporting_discovery_ids\": [13, 29, 40]}\n    ],\n    \"complexes\": [],\n    \"partners\": [\n      \"SGK1\",\n      \"YWHAB\",\n      \"SCNN1A\",\n      \"SLC12A3\",\n      \"GRK2\",\n      \"NDFIP1\",\n      \"AMOTL1\",\n      \"USP7\"\n    ],\n    \"other_free_text\": []\n  }\n}","audit_flag":null,"evaluation":{"pairwise":"win","faith_supported":7,"faith_total":7,"faith_pct":100.0}}